nih public access free radic biol med a,*,1 b,1 c,* b,2 … · 2016. 6. 6. · free radic biol med....
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Cannabidiol protects liver from binge alcohol-induced steatosisby mechanisms including inhibition of oxidative stress andincrease in autophagy
Lili Yanga,*,1, Raphael Rozenfeldb,1, Defeng Wub, Lakshmi A. Devib, Zhenfeng Zhangc,*, andArthur Cederbaumb,2
Lili Yang: [email protected]; Zhenfeng Zhang: [email protected]; Arthur Cederbaum:[email protected] of Public Health, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
bMount Sinai School of Medicine, New York, NY 10029, USA
cState Key Laboratory of Oncology in South China, Collaborative Innovation Center for CancerMedicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China
Abstract
Acute alcohol drinking induces steatosis, and effective prevention of steatosis can protect liver
from progressive damage caused by alcohol. Increased oxidative stress has been reported as one
mechanism underlying alcohol-induced steatosis. We evaluated whether cannabidiol, which has
been reported to function as an antioxidant, can protect the liver from alcohol-generated oxidative
stress-induced steatosis. Cannabidiol can prevent acute alcohol-induced liver steatosis in mice,
possibly by preventing the increase in oxidative stress and the activation of the JNK MAPK
pathway. Cannabidiol per se can increase autophagy both in CYP2E1-expressing HepG2 cells and
in mouse liver. Importantly, cannabidiol can prevent the decrease in autophagy induced by
alcohol. In conclusion, these results show that cannabidiol protects mouse liver from acute
alcohol-induced steatosis through multiple mechanisms including attenuation of alcohol-mediated
oxidative stress, prevention of JNK MAPK activation, and increasing autophagy.
Keywords
Alcohol; Steatosis; Cannabidiol; Oxidative stress; Autophagy; Free radicals
Both acute and chronic alcohol drinking induces hepatosteatosis [1,2]. Although steatosis is
a reversible injury to liver, its progression can develop into more severe liver problems such
as hepatitis and cirrhosis. Recent research showed that oxidative stress is an important factor
contributing to mechanisms that induce steatosis [2–4]. Although exact pathways for this
have not been clearly identified or studied in detail, possible mechanisms may include
reactive oxygen activation of lipogenic transcription factors, e.g., SREBP, or decline in
lipolytic factors, e.g., PPARα, or mitochondrial dysfunction and decline in fatty acid
*Corresponding authors.1These authors contributed equally to this work.2Fax: +212 796 8214.
NIH Public AccessAuthor ManuscriptFree Radic Biol Med. Author manuscript; available in PMC 2014 September 01.
Published in final edited form as:Free Radic Biol Med. 2014 March ; 68: 260–267. doi:10.1016/j.freeradbiomed.2013.12.026.
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oxidation, or activation of the unfolded protein response and endoplasmic reticulum stress.
CYP2E1, a cytochrome P450 enzyme, has been shown to play a major role in alcohol-
induced oxidative stress and fat accumulation as well as non-alcohol-induced steatosis [1–
4]. Studies with CYP2E1-knockout mice showed little or no oxidative stress produced by
alcohol compared to CYP2E1-overexpressing mice [1]. Increased oxidative stress was also
found in CYP2E1-overexpressing HepG2 cells compared to HepG2 cells lacking CYP2E1
expression [4,5]. These data indicate that oxidative stress is important for the induction of
steatosis by alcohol and suggest that compounds that could effectively prevent the induction
of oxidative stress represent potential drugs for the prevention of liver steatosis caused by
alcohol drinking.
Cannabidiol (CBD) is a nonpsychoactive molecule from the cannabis plant that exhibits
unique pharmacological and biological activities. In recent years, CBD has been shown to
act as an anti-inflammatory molecule, to be able to block the progression of arthritis [6] and
type 1 diabetes [7], to have antioxidant actions [8], and to act as an antiproliferative
compound inducing a loss in viability in various tumor cell lines [9–11]. However, the
molecular mechanisms mediating the biological effects of CBD are not well understood.
Given the antioxidant properties of CBD, we hypothesized that this compound could exhibit
therapeutic properties in the context of alcohol-induced liver injury, by alleviating oxidative
stress-induced hepatocellular injury and fat accumulation.
Cannabinoid compounds have also been shown to induce autophagy [12,13]. This is of
particular interest in the context of liver injury, as autophagy has recently emerged as a
major regulator of lipid homeostasis in the liver [14–18]. Autophagy, especially
macroautophagy, is a mechanism by which hepatocytes break down lipids (a mechanism
called lipophagy). Inhibition of macroautophagy causes lipid droplets to increase in size and
number and to accumulate in hepatocytes. A decreased rate of lipolysis leads to an increase
in cellular triglyceride (TG) content, a feature observed in steatosis. In some studies, acute
alcohol was shown to decrease autophagy in the liver, leading to steatosis [2,19]. However,
other studies showed that acute and chronic alcohol increased autophagy, possibly to protect
against alcohol-induced fat accumulation [20,22]. In line with these observations and the
proautophagic properties of cannabinoids, we determined whether CBD can protect against
alcohol-induced liver steatosis through induction of autophagy.
Materials and methods
Materials
CBD was obtained from Tocris Bioscience (Ellisville, MO, USA). Reactive oxygen species
(ROS) detection kit was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA).
ATP determination kit was from Molecular Probes (Eugene, OR, USA). Antibodies against
phospho-c-Jun N-terminal kinase (pJNK), JNK, microtubule-associated protein 1A/1B light-
chain 3 (LC3), pP38 mitogen-activated protein kinase (MAPK), and P38 MAPK were
obtained from Cell Signaling (Danvers). Antibody against CYP2E1 was a generous gift
from Dr. Jerry Lasker.
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Mouse acute alcohol model
Animal experiments were performed with the approval of the Mount Sinai Animal Care and
Use Committee. C57Bl/6 mice weighing 25–30 g, at 8–10 weeks of age (purchased from
The Jackson Laboratory), were gavaged with ethanol (30% v/v in saline, 4 g/kg, every 12 h)
for 5 days. CBD (5 mg/kg, every 12 h) or vehicle (Tween 80 2%–saline) was injected
intraperitoneally (ip) 30 min before each ethanol gavage. After the stated treatment period,
mice were anesthetized by ip administration of 50 mg/kg pentobarbital, and serum and liver
were collected. Hematoxylin and eosin (H&E) and oil red O staining of liver slices was
performed as previously described [2,4,5]. Paraffin slides were deparaffinized and blocked
as previously reported [20]. Antibodies against 4-hydroxynonenal (4-HNE), 3-nitrotyrosine
(3-NT), and pJNK were added at 1:200 dilution and incubated at 4°C overnight. Slides were
washed with phosphate-buffered saline and stained with the Histostain Plus Broad Spectrum
(DAB) kit. Images were acquired under the light microscope at 200 × magnitude. The oil red
O staining was quantified by ImageJ software from the U.S. National Institutes of Health.
ATP assay
ATP content was determined using an ATP Determination Kit (A22066, Molecular Probes).
Each reaction contained 1.25 μg/ml firefly luciferase, 50 μM D-luciferin, and 1 mM
dithiothreitol in 1 × reaction buffer. Cells were treated with vehicle or CBD or ethanol or
CBD plus ethanol. After a 15-min incubation with 1 mg liver protein, luminescence was
measured in a luminometer. Results are expressed as arbitrary units of luminescence
compared to that of the vehicle-treated control cells, which was taken as 100 arbitrary units.
Triglyceride analysis
TG levels in the liver lysates were analyzed following the instructions in the TG analysis kit
from Pointe Scientific (Canton, MI, USA). TGs in the sample are hydrolyzed by lipase to
glycerol and fatty acids. The glycerol is then phosphorylated by ATP to glycerol 3-
phosphate. Glycerol 3-phosphate is then converted to dihydroxyacetone phosphate and
hydrogen peroxide by glycerophosphate oxidase. The hydrogen peroxide then reacts with 4-
aminoantipyrine and 3-hydroxy-2,4,6-tribomobenzoic acid in a reaction catalyzed by
peroxidase to yield a red quinoneimine dye. The intensity of the color produced is directly
proportional to the concentration of triglycerides in the sample when measured at 540 nm.
Oil red O and ROS assays
E47 cells, which express CYP2E1 [21], were incubated with 50 or 100 mM ethanol for 5
days (fresh medium with ethanol was added every 24 h) in the absence or presence of CBD
(2 and 5 μM). Oil red O staining and ROS detection by flow cytometry of E47 cells were
performed as previously described [2,4,5,19]. Total ROS detection was assayed using the
Total ROS Detection Kit (Enzo Life Sciences) which contains a nonfluorescent, cell-
permeative ROS detection dye. The dye can react directly with a wide range of reactive
species, such as hydrogen peroxide, peroxynitrite, and hydroxyl radicals, to yield a green
fluorescent product indicative of cellular production of various reactive oxygen or nitrogen
species. Upon staining, the fluorescent product generated can be visualized using a flow
cytometer equipped with a blue (488 nm) laser. The shift of fluorescence peaks to the right
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is indicative of increased ROS production. Flow cytometric analyses were performed on a
three-laser LSRII with DiVa software (BD Biosciences). Data were analyzed with FlowJo
software. Activity of CYP2E1 was assayed by the oxidation of p-nitrophenol [2,5].
Western blot analysis
Levels of CYP2E1, pJNK, JNK, LC3, pP38 MAPK, and P38 MAPK in 20–100 μg of
protein samples from freshly prepared liver homogenate fractions were determined using
Western blot analysis. Blots were scanned using an Odyssey Imaging System (Li-Cor
Biosciences, Lincoln, NE, USA). All specific bands were quantified with the Automated
Digitizing System (ImageJ programs, version 1.34 S, National Institutes of Health). Results
are shown for one experiment but quantification is from three separate experiments.
LC3 and autophagy assay
LC3 is microtubule-associated protein 1 A/1B light-chain 3, a soluble protein with a
molecular mass of approximately 17 kDa. It is distributed ubiquitously in mammalian
tissues and cultured cells. During autophagy, a cytosolic form of LC3 (LC3-I) is conjugated
to phosphatidylethanolamine (lipidation) to form an LC3–phosphatidylethanolamine
conjugate (LC3-II), which is recruited to autophagosomal membranes. The lipidated form of
LC3 is referred to LC3-II, whereas LC3-I is not lipidated. The LC3-II/LC3-I ratio is often
used to assay autophagy and, when assayed in the presence of lysosomal inhibitors such as
chloroquine, it represents autophagic flux.
Statistical analysis
Values reflect means ± SD. One-way ANOVA with subsequent post hoc comparisons by
Tukey HSD were performed by SPSS analysis software (version 10.0). A p value of less
than 0.05 was considered statistically significant and results are from experiments using four
to six mice or six replicates of each group of cells.
Results
CBD decreases ethanol-induced liver injury
To assess the effect of CBD on ethanol-induced hepatotoxicity, we induced liver injury by
treating mice with ethanol (30% v/v in saline, 4 g/kg, every 12 h for 5 days). CBD (5 mg/kg,
every 12 h) or vehicle was injected ip 30 min before ethanol gavage. Ethanol gavage led to
an increase in serum aspartate aminotransferase (AST) that was prevented by CBD (Fig.
1A). Ethanol gavage produced a 60% decrease in hepatic ATP levels (Fig. 1C), suggestive
of hepatic bioenergetic injury. This decline in ATP was completely reversed by CBD (Fig.
1C). Ethanol gavage led to a 60% elevation in hepatic triglycerides. This effect was
significantly attenuated by CBD treatment (Fig., 1B). The CBD treatment also lowered basal
TG levels. These effects of CBD were accompanied by a prevention of ethanol-induced
steatosis as demonstrated by H&E (Fig. 2A) and oil red O staining of liver sections (Fig.
2B).
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CBD reduces ethanol-induced oxidative stress in livers of ethanol-treated mice
We then examined if CBD was attenuating liver injury by acting as an antioxidant molecule.
Liver sections were treated with anti-4-HNE and anti-3-NT IgG to detect 4-HNE or 3-NT
adducts (Figs. 3A and B). Ethanol led to an increase in 4-HNE staining. Interestingly, this
effect was attenuated in livers from mice treated with CBD (Fig. 3A, images 3 and 4). There
was, however, no change in 3-NT protein adducts by ethanol, or CBD, or ethanol/CBD (Fig.
3B).
CBD treatment prevents JNK activation
Binge ethanol has been shown to activate the JNK and P38 MAPK pathways [2,22–24].
Inhibition of MAPK partially prevents alcohol- and other xenobiotic-mediated liver injury
[2,25,26]. We examined the effect of CBD on ethanol-induced JNK and P38 MAPK
phosphorylation. Staining of liver sections with anti-pJNK IgG indicated that CBD
prevented JNK activation by binge ethanol (Fig. 4A). Western blot analyses of liver
homogenates confirmed that ethanol induced the phosphorylation of JNK1 about fourfold
and that CBD treatment partially blocked this increase in JNK1 phosphorylation (Fig. 4B).
The binge ethanol treatment had no effect on activation of P38 MAPK (Fig. 4C).
CBD reduces ethanol-induced oxidative stress in CYP2E1-expressing HepG2 cells
To confirm the antioxidant action of CBD as an explanation for its effect on ethanol-induced
liver injury, we examined the effect of CBD on ethanol-induced oxidative stress in vitro.
HepG2 cells stably expressing CYP2E1 (E47 cells) were used for this purpose. We have
previously demonstrated the requirement of CYP2E1 activity to facilitate ethanol toxicity in
these cells [4,5]. E47 cells were incubated with 50 or 100 mM ethanol for 5 days, in the
absence or presence of CBD (5 μM). Ethanol treatment led to increases in ROS levels that
were decreased by cotreatment with CBD (Fig. 5A). Quantification of the areas in Fig. 5A
indicated that there were 2.5- and 3.5-fold increases produced by 50 and 100 mM ethanol,
respectively, and that CBD lowered these to increases of about 10–20% (data not shown). In
addition we observed that treatment with CBD also reduced the basal levels of ROS
produced by CYP2E1 in the E47 cells (Fig. 5A, graph 1). CBD treatment also decreased the
ethanol-induced increase in the TG content in these cells (Fig. 5B) and decreased lipid
droplet accumulation induced by ethanol as shown by oil red O staining (Figs. 5C and D).
CBD effects were not due to changes in CYP2E1 protein levels or activity in the E47 cells
(Figs. 5E and F). These results in a well-established model to study the effects of alcohol
metabolism on cellular injury are consistent with the ability of CBD to mitigate ethanol-
induced liver injury in vivo.
CBD stimulates autophagy in vitro and in vivo
Several recent studies have demonstrated the importance of autophagy in hepatocyte
homeostasis. In particular, growing evidence indicates that stimulation of autophagy
decreases hepatocyte oxidative stress and fat accumulation by clearing damaged
mitochondria and lipid droplets from hepatocytes [27–29]. We examined the possibility that
one mechanism by which CBD exerted its protective effects against ethanol toxicity was by
stimulating autophagy. During autophagy, LC3-I (cytosolic form of LC3) is conjugated to
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phosphatidylethanolamine (lipidation) to form LC3-II (LC3–phosphatidylethanolamine
conjugate), which is recruited to autophagosomal membranes. The lipidated form of LC3 is
referred to as LC3-II, whereas LC3-I is not lipidated. The LC3-II/LC3-I ratio is often used to
assay autophagy and when assayed in the presence of lysosomal inhibitors such as
chloroquine, it assays autophagic flux. LC3 lipidation was evaluated as an index of
autophagy by Western blot analysis. Treatment of E47 cells with CBD led to an increase in
levels of LC3-II, indicating an increase in autophagosome formation. This effect was further
increased upon cotreatment with chloroquine, confirming an induction of autophagy by
CBD (Fig. 6A). We next examined autophagy in livers from the ethanol-treated mice.
Ethanol decreased autophagy, as indicated by a decrease in LC3-II levels and in the LC3-II/
LC3-I ratio (Fig. 6B). This inhibition of autophagy by ethanol was not seen in livers from
mice that received CBD plus ethanol (Fig. 6B), indicating that CBD prevents alcohol-
mediated inhibition of autophagy in vivo. Both in vitro and in vivo experiments showed that
CBD promotes autophagy, which could play a role in the mechanisms by which CBD
protects liver from acute alcohol-induced steatosis.
Discussion
Acute alcohol drinking induces liver steatosis [2,19,27,29]. The induction of liver steatosis
is promoted by CYP2E1, as wild-type mice with CYP2E1 expression displayed steatosis,
but CYP2E1-knockout mice exhibit strongly decreased steatosis after acute alcohol
treatment [1,2,19]. In the current study, lipid accumulation was found in CYP2E1-
expressing HepG2 cells after ethanol treatment, and liver steatosis was observed in mice
treated with acute alcohol. Mechanisms by which CYP2E1 promotes ethanol induction of
steatosis include elevated ROS, activation of MAPKs such as JNK, inhibition of autophagy,
and decreases in levels of PPARα, a major regulator of fatty acid oxidation [1,2]. Oxidative
stress has been reported to be one major cause of liver steatosis by alcohol. Antioxidants
such as N-acetylcysteine attenuate acute alcohol-induced steatosis [2,30]. We previously
found that a JNK inhibitor partially blocked acute ethanol-induced steatosis [2]. CBD is a
nonpsychotic cannabinoid, which has been shown to have anti-inflammatory and antioxidant
properties. CBD has also been reported to function as an antioxidant in preventing glutamate
toxicity and preventing neurotoxicity by acute alcohol [31]. Consistent with reports that
CBD has antioxidant activity, we found that CBD can reduce ROS production in HepG2
cells expressing CYP2E1 and that CBD inhibited the increase in ROS induced by alcohol
treatment. CBD protected liver from acute alcohol-induced steatosis and lowered 4-HNE
levels via its antioxidant property. CBD did not alter CYP2E1 levels or catalytic activity;
therefore the CBD prevention of alcohol-induced steatosis and oxidant stress is not due to
effects on CYP2E1.
How activation of CYP2E1 by alcohol consumption, in addition to oxidative stress,
promotes steatosis requires further mechanistic evaluation. The JNK MAPK pathway is a
major signaling pathway, which initiates apoptosis and cell death [32,33]. Recent reports
also showed that activation of the JNK MAPK pathway by alcohol may be a key factor
regulating the increase in liver steatosis, because inhibition of JNK activation prevented
acute alcohol-induced steatosis [2]. We found that CBD can inhibit the increase in JNK
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MAPK phosphorylation by acute alcohol, which may be due to its antioxidant actions.
Inhibition of JNK activation may contribute to CBD's anti-steatosis effects.
While a basal level of autophagy is required for the survival of cells or organisms, prolonged
activation of autophagy may have adverse effects. In mammalian systems, autophagy is
stimulated by nutrient starvation or deprivation of growth factors [34–38]. Although it is not
clear yet whether alcohol increases or decreases autophagy under different conditions, it is
consistent that autophagy is a protective mechanism against alcohol-induced liver injury
[2,19,27,29]. Increasing autophagy can protect cells from injury by various stimuli, as
inhibition of autophagy increases toxicity to cells. Genetically enhancing autophagy by
overexpressing Atg7 could alleviate hepatic steatosis induced by a high-fat diet [39].
Carbamazepine, an FDA-approved antiepileptic drug, can alleviate fatty liver by inducing
autophagy [40]. In contrast, loss of autophagy in vitro or in vivo increases lipid
accumulation in cells and in liver [2,4,19]. We found that CBD can stimulate autophagy in
HepG2 cells that express CYP2E1. The increased flux from LC3-I to LC3-II was confirmed
by treating cells with a lysosome inhibitor. Because the LC3-II/LC3-I ratio is used to assay
autophagy, the increased flux from LC3-I to LC3-II indicates increases in autophagy. CBD-
mediated increase in autophagy may be important for the prevention of alcohol-induced
steatosis by CBD.
In conclusion, this is the first report demonstrating that CBD can alleviate lipid
accumulation in both an in vitro HepG2 cell model and an in vivo binge alcohol treatment
model by multiple mechanisms. These mechanisms may involve activation of autophagy,
inhibition of the JNK MAPK pathway, and inhibition of oxidative stress, and all three
mechanisms could be important for alleviating steatosis.
Acknowledgments
This work was supported by Grants AA-018790 and AA-021362 from the NIAAA (to A.I.C.) and NIH Grant DA008863 (to L.A.D.).
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Fig. 1.Binge ethanol induces liver injury and accumulation of lipids in mice, and CBD reverses
these effects. (A) Serum AST levels; (B) TG levels in liver; (C) ATP levels in liver. Results
are from four to six mice in each group. *p < 0.05 and **p < 0.01.
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Fig. 2.(A) H&E staining and (B) oil red O staining showing increased lipid accumulation in mouse
liver after binge alcohol treatment. CBD decreases this lipid accumulation (image 4
compared to image 3 in (A) and (B)).
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Fig. 3.(A) Binge alcohol drinking increases 4-HNE staining in mouse liver (image 3 compared to
image 1), and CBD reverses this increase (image 4 compared to image 3). Black arrows
point to areas of positive staining of 4-HNE. (B) Binge alcohol, CBD, or ethanol/CBD do
not change 3-NT staining in mouse liver (image 2, 3, or 4 compared to image 1).
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Fig. 4.Binge alcohol drinking increases phosphorylation of c-Jun N-terminal kinase (JNK) as
shown by (A) immunohistochemistry (image 3 compared to image 1) and (B) Western blot.
Livers were collected 18 h after the last ethanol gavage. CBD partially blocked the
activation of JNK by binge alcohol. *p < 0.05 comparing ethanol/vehicle to vehicle/vehicle.
&p < 0.05 comparing ethanol/CBD to ethanol/vehicle. (C) P38 MAPK phosphorylation is
not increased by binge alcohol consumption.
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Fig. 5.(A) In cytochrome P450 2E1 (CYP2E1)-expressing HepG2 cells, either 50 or 100 mM
ethanol treatment increased ROS production (graphs 2 and 4), and CBD partially prevented
the increase in ROS (graphs 3 and 5 compared to 2 and 4). ROS were assayed using the
Total ROS Detection Kit. The distribution of fluorescence intensity is shown as a histogram,
with the y axis indicating the percentage of cells in the total population displaying the
specific fluorescence intensity shown on the x axis. (B and C) In vitro 100 mM ethanol
treatment increased lipid accumulation in CYP2E1-expressing HepG2 cells. (B) TG levels,
expressed as mg/mg protein; *p < 0.05, **p < 0.01. (C) Oil red O staining showing that
CBD prevents this lipid accumulation. (D) Quantification of oil red O staining was carried
out using the ImageJ program. Staining in the vehicle/vehicle group was taken as 100, and
staining of all other groups was expressed relative to the vehicle/vehicle group. *p < 0.05
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compared to vehicle/vehicle group. (E and F) CYP2E1 protein expression and catalytic
activity were assayed 48 h after the last treatment with CBD. CBD itself does not have any
effect on either CYP2E1 protein expression or CYP2E1 activity.
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Fig. 6.Effects of CBD and CBD + ethanol on autophagy. LC3 is microtubule-associated protein 1
A/1B light-chain 3. During autophagy, a cytosolic form of LC3 (LC3-I) is conjugated to
phosphatidylethanolamine (lipidation) to form LC3–phosphatidylethanolamine (LC3-II),
which is recruited to autophagosomal membranes. The LC3-II/LC3-I ratio is often used to
assay autophagy and, when assayed in the presence of lysosomal inhibitors such as
chloroquine, it indicates autophagic flux. (A) E47 HepG2 cells were incubated with 5 μM
CBD in the absence or presence of 10 mM chloroquine (CQ) for up to 8 h and immunoblots
were carried out to assay for LC3-II, LC3-I, and actin. (B) Binge alcohol drinking decreases
autophagy in mouse liver. Pretreatment of mice with 5 mg/kg CBD prevents the decrease in
autophagy by alcohol. *p < 0.05 compared to vehicle/vehicle group. #p < 0.05 compared to
ethanol/vehicle group. &p < 0.05 compared to vehicle/CBD group.
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